A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis Jian-Kui Du PhD1,2a, Qing Yu M.S2a, Yu-Jian Liu PhD3, Shu-Fang Du M.S2, Li-Yang Huang M.Sc2, Dan-Hong Xu M.Sc3, Xin Ni PhD1,2*, Xiao-Yan Zhu PhD 2* 1National Clinical Research Center for Geriatric Disorders and National International Joint Research Center for Medical Metabolomics, Xiangya Hospital, Central South University, Changsha, Hunan, China; 2Department of Physiology, Navy Medical University, Shanghai, China; 3School of Kinesiology, Shanghai University of Sport, Shanghai, China aThese authors contributed equally to this work and should be considered as co-first authors Short title: KLK8 promotes EndMT in diabetic cardiomyopathy *Corresponding Authors: Xiao-Yan Zhu, MD, PhD, Navy Medical University, 800 Xiangyin Rd, Shanghai, 200433, China. Email: [email protected]; Xin Ni, MD, PhD, National International Joint Research Center for Medical Metabolomics, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008, China. Email: [email protected]. Abstract Rationale: Among all the diabetic complications, diabetic cardiomyopathy, which is characterized by myocyte loss and myocardial fibrosis, is the leading cause of mortality and morbidity in diabetic patients. Tissue kallikrein-related peptidases (KLKs) are secreted serine proteases, that have distinct and overlapping roles in the pathogenesis of cardiovascular diseases. However, whether KLKs are involved in the development of diabetic cardiomyopathy remains unknown.The present study aimed to determine the role of a specific KLK in the initiation of endothelial-to-mesenchymal transition (EndMT) during the pathogenesis of diabetic cardiomyopathy. Methods and Results-By screening gene expression profiles of KLKs, it was found that KLK8 was highly induced in the myocardium of mice with streptozotocin-induced diabetes. KLK8 deficiency attenuated diabetic cardiac fibrosis, and rescued the impaired cardiac function in diabetic mice. Small interfering RNA (siRNA)-mediated KLK8 knockdown significantly attenuated high glucose-induced endothelial damage and EndMT in human coronary artery endothelial cells (HCAECs). Diabetes-induced endothelial injury and cardiac EndMT were significantly alleviated in KLK8-deficient mice. In addition, transgenic overexpression of KLK8 led to interstitial and perivascular cardiac fibrosis, endothelial injury and EndMT in the heart. Adenovirus-mediated overexpression of KLK8 (Ad-KLK8) resulted in increases in endothelial cell damage, permeability and transforming growth factor (TGF)-β1 release in HCAECs. KLK8 overexpression also induced EndMT in HCAECs, which was alleviated by a TGF-β1-neutralizing antibody. A specificity protein-1 (Sp-1) consensus site was identified in the human KLK8 promoter and was found to mediate the high glucose-induced KLK8 expression. Mechanistically, it was identified that the vascular endothelial (VE)-cadherin/plakoglobin complex may associate with KLK8 in HCAECs. KLK8 cleaved the VE-cadherin extracellular domain, thus promoting plakoglobin nuclear translocation. Plakoglobin was required for KLK8-induced EndMT by cooperating with p53. KLK8 overexpression led to plakoglobin-dependent association of p53 with hypoxia inducible factor (HIF)-1α, which further enhanced the transactivation effect of HIF-1α on the TGF-β1 promoter. KLK8 also induced the binding of p53 with Smad3, subsequently promoting pro-EndMT reprogramming via the TGF-β1/Smad signaling pathway in HCAECs. The in vitro and in vivo findings further demonstrated that high glucose may promote plakoglobin-dependent cooperation of p53 with HIF-1α and Smad3, subsequently increasing the expression of TGF-β1 and the pro-EndMT target genes of the TGF-β1/Smad signaling pathway in a KLK8-dependent manner. Conclusions: The present findings uncovered a novel pro-EndMT mechanism during the pathogenesis of diabetic cardiac fibrosis via the upregulation of KLK8, and may contribute to the development of future KLK8-based therapeutic strategies for diabetic cardiomyopathy. Keywords: KLK8; endothelial-to-mesenchymal transition; cardiac fibrosis; diabetic cardiomyopathy; plakoglobin Graphical Abstract Introduction The global incidence of diabetes mellitus has emerged as a major threat to worldwide health [1,2]. Uncontrolled diabetes leads to a number of complications, including diabetic cardiomyopathy [3], nephropathy [4], and vision problems [5]. Among all the diabetic complications, diabetic cardiomyopathy, which is characterized by myocyte loss and myocardial fibrosis, is the leading cause of mortality and morbidity in diabetic patients [3,6,7]. Progressive cardiac fibrosis has been found in diabetic patients and in animal models of diabetes [7,8]. Excessive collagen deposition in the myocardium reduces cardiac compliance, thus contributing to increased left ventricular stiffness and impaired contractile function, and ultimately heart failure [7,8]. Despite its clinical significance, the pathological basis of diabetes-associated cardiac fibrosis remains largely unclear. Myofibroblasts are major contributors to extracellular matrix (ECM) accumulation in fibrotic disease, and are known to be derived from resident fibroblasts, epithelial cells, and bone marrow-derived cells [9]. As the initial targets of hyperglycemic damage, endothelial cells play a major role in the production of ECM proteins in all chronic diabetic complications [10]. A result of sustained endothelial injury during diabetes mellitus is that endothelial cells undergo a process of transdifferentiation of endothelial cells into mesenchymal cells called endothelial-to-mesenchymal-transition (EndMT), which further switches their phenotype to myofibroblasts [11,12]. Of note, EndMT is considered to be an important mechanism of diabetic cardiac fibrosis [13,14]. However, the factors promoting EndMT and cardiac fibrosis during the development of diabetic cardiomyopathy require to be further elucidated. The tissue kallikrein-related peptidase (KLK) family is a group of secreted serine proteases encoded by tandemly arranged genes of multigene families, which comprises 15 genes in human [15]. The implication of KLKs in the development of fibrotic diseases and stabilization of endothelial cells has attracted considerable attention, since KLK family members are deeply involved in the degradation of ECM proteins such as fibronectin and laminin [16,17]. Among KLKs, KLK1 is the member most widely studied, and confers protection against endothelial dysfunction and cardiac fibrosis induced by diabetes [18]. However, the opposite effects are observed with other KLKs family members. For example, overexpression of KLK8 can eventually lead to cardiac hypertrophy and fibrosis [19]. Notably, a combination of serine proteases including KLKs 1, 5 and 6, and elastases 1 and 2, can induce EndMT in human aortic endothelial cells [20]. These studies indicate that KLK family members have distinct and overlapping roles in the pathogenesis of cardiovascular diseases. By screening the mRNA expression of the KLK family in the myocardium, it was found that KLK8 was the highest induced KLK member in diabetic myocardium. Using both KLK8 knockout mice and KLK8 transgenic rats, the present study investigated the precise role of KLK8 in mediating diabetic cardiomyopathy and demonstrated that upregulation of KLK8 contributes to the development of EndMT and diabetic cardiac fibrosis. In addition, the molecular mechanisms underlying KLK8-mediated EndMT and cardiac fibrosis in the context of diabetes were illustrated. Results KLK8 expression is significantly increased in diabetic myocardium To investigate the role of KLKs in the development of diabetes-associated cardiomyopathy, the present study firstly examined the mRNA expression levels of KLKs in heart tissues obtained from streptozotocin (STZ)-induced diabetic mice at 12 and 24 weeks after the induction of diabetes mellitus. As shown in Figure 1A, KLK6, KLK8 and KLK12 were significantly upregulated, while KLK1, KLK5, KLK7 and KLK11 were downregulated in the heart tissues of STZ-induced diabetic mice compared with the levels exhibited by age-matched non-diabetic controls. Among all the upregulated KLK family members, KLK8 was the highest induced KLK. Expression of KLK8 in diabetic myocardium was then determined by immunohistochemistry staining and western blotting. As shown in Figure 1B-C, KLK8 staining was significantly increased in both cardiomyocytes and coronary endothelial cells in the myocardium of diabetic mice compared with the expression levels exhibited by the control group. As expected, Masson’s trichrome staining revealed a significant collagen deposition in both interstitial and perivascular regions in the diabetic myocardium (Figure 1D-E). Immunoblotting also confirmed the induction of KLK8 protein expression in the myocardium of diabetic mice (Figure 1F). KLK8 deficiency attenuates diabetic cardiac fibrosis Mice with global deletion of KLK8 were then used to investigate whether KLK8 deficiency affects diabetes-associated cardiac fibrosis. As shown in Figure 2A, the diabetes-induced upregulation of cardiac KLK8 was blunt in KLK8-deficient (KLK8-/-) mice. STZ-induced low insulin levels and hyperglycemia occurred in both KLK8-/- and KLK8+/+ mice, whereas the levels of insulin and blood glucose in non-diabetic mice were normal (Table 1, Figure S1). Under baseline conditions, KLK8-/- mice exhibited similar levels of body weight, total cholesterol (TC), triglyceride (TG),